原发性胆汁性肝硬化模型中淋巴细胞增殖及活化诱导凋亡机制研究  

Lymphocyte proliferation and activation induced cell death in model of primary biliary cirrhosis

作　　者：蒋廷旺[1] 熊怀民[1] 盛建华[1] 龚燕萍[1] 沈艳红[1] 陆建文[1] 许国华[1] 邓安梅[2] 仲人前[2] 

机构地区：[1]江苏省常熟市第二人民医院检验科,215500 [2]上海长征医院实验诊断科全军临床免疫中心

出　　处：《中华消化杂志》2010年第4期217-221,共5页Chinese Journal of Digestion

基　　金：国家863计划资助项目（2006AA02Z496）

摘　　要：目的通过检测原发性胆汁性肝硬化（PBC）动物模型肝脏和脾脏淋巴细胞增殖及活化诱导凋亡（AICD），了解PBC模型小鼠的免疫耐受情况。方法聚肌苷酸胞嘧啶核苷酸（polyI：C）5mg／kg剂量注射C57BL／6小鼠建立PBC模型，分离肝、脾淋巴细胞并纯化CD4^＋T淋巴细胞，然后以M2蛋白及刀豆蛋白A（ConA）结合抗-CD3刺激细胞增殖及AICD，定量PCR和免疫印迹技术检测凋亡相关基因及信号蛋白。结果①M2蛋白刺激后，空白对照组和PBS组的细胞增殖能力（分别为0．1988±0．0111和0．2068±0．0115）差异无统计学意义（P〉0．05），但是PBC组小鼠细胞增殖能力（0．358±0．022）与以上两组相比显著增强，且PBC组小鼠肝内淋巴细胞增殖能力强于脾淋巴细胞（P〈0．01）。②空白对照组和PBS组之间AICD情况差异无统计学意义（74．70％±4．58％比74．20％±4．44％，P〉0．05），但均显著高于PBC组（44．85％±6．47％，P〈0．01），同时PBC组小鼠肝内CD4^＋T细胞的凋亡率显著低于脾脏（P〈0．01）。③PBC组小鼠肝脾淋巴细胞FasL、TRAIL基因的表达较PBS组均明显降低（P〈0．01），而Fas表达无明显改变。④PBC组小鼠CD4^＋T细胞长构型Fas相关死亡区域样白细胞介素-1G转化酶抑制蛋白（FLIPL）表达明显升高，且肝内T细胞表达较脾脏显著增加（P〈0．01）。结论FLIPL表达增高可能是AICD受到抑制的重要原因，同时FasL和TRAIL mRNA表达降低可能也起到一定的作用。Objective To investigate the immune tolerance in animal models of primary biliary cirrhosis （PBC） by determining the cell proliferation and activation induced cell death （AICD）. Methods C57BL/6 mice were injected with 5 mg/kg of polyI： C to develope PBC models. The lymphocytes and CD4^＋ T cells were separated from spleens and livers 16 weeks later and were stimulated by M2, conA and anti-CD3 for cell proliferation and AICD. Expression of apoptosis related genes and proteins were detected by real time polymerase chain reaction （PCR） and Western blotting, respectively. Results （3） The lymphocyte proliferation was 0. 1988 ± 0. 0111 in blank controls and 0. 2068±0. 0115 in PBS treated mice with no significant difference （P〉0. 05）. However, an abundant lymphocyte proliferation was found in PBC mice （0. 358 ± 0. 022）, which was higher than that in controls and PBS treated mice. The proliferation of lymphocyte from liver was greater than that from spleen in PBC mice （P〈0.01）. （2） The apoptotic rate in blank controls （74.70%±4.58%） and PBS treated mice （74.20%±4.44%） was higher than that in PBC mice （44.85% ±6.47%, P〈0. 01）, but no difference was found between blank controls and PBS treated mice （P〉0.05）. Furthermore, the apoptosis rate of T cells from livers were significantly lower than that from spleens in PBC mice （P〈0. 01）. ③ The expressions of FasL and tumor necrosis factor related apoptosis-indueing ligand （TRAIL） in PBC mice were lower than those in PBS treated mice （P〈0. 01）, but there was no change in expression of Fas was found. ④ The expression of Fas-associated death domain-like interleukin-l-β-converting enzyme-inhibitory protein （FLIPL） in PBC mice was higher than that in blank controls. Moreover, the expression of FLIPL in livers was higher than that in spleens in PBC mice （P〈0. 01）. Conclusions The elevated expression of FLIPL may inhibit AICD. Besides, the decreased expressions of FasL and TRAIL may also hel

关 键 词：原发性胆汁性肝硬化 凋亡 免疫耐受 

分 类 号：R575.2[医药卫生—消化系统]